Polymorphonuclear leukocytes, neutrophils, are the most abundant leukocytes in peripheral blood. They play a major role in innate immunity and may contribute to the development of the adaptive immune response. The early and selective recruitment of neutrophils into inflamed tissues is thought to be a major factor in the tissue damage/destruction seen in a number of disease states. The chemokines Interleukin-8 (IL8) and Growth-Regulated Oncogene alpha (GROalpha) control much of the normal function of neutrophils. These chemokines are also implicated in the diseases associated with neutrophil dysfunction. Research is beginning to investigate the specific signal transduction pathways activated by these chemokines to regulate neutrophil functions. Our long-range goal is to define the signal transduction mechanisms used by chemokines to activate neutrophils in order to develop effective therapeutic strategies to regulate this activation. Polarization is the first step in the process of neutrophil migration into sites of inflammation. However, the signal transduction pathways that regulate chemokine stimulated neutrophil polarization remain undefined. The objective of this proposal is to determine the mechanisms that induce chemokine stimulated neutrophil polarization. The central hypothesis of this proposal is that IL8, in contrast to GROalpha regulates neutrophil polarization independent of phosphatidylinositol-3 (PI3) kinase because IL8 stimulates a more robust activation of several different signal transduction molecules. This hypothesis is based on strong preliminary data that suggest that efficient activation of Ras by IL8, in contrast to GROalpha allows for PI3 kinase independent regulation of neutrophil polarization, because IL8 binds to both CXCR1 and CXCR2. The rationale for the proposed research is that once the mechanisms controlling neutrophil polarization are defined, effective therapies can be developed to control this process. The central hypothesis will be tested and the objective accomplished through two specific aims: 1) identify the signal transduction molecule that allows IL8 to by-pass PI3 kinase in the regulation of neutrophil polarization; 2) determine the extent to which the chemokine receptors CXCR1 and CXCR2 contribute to PI3 kinase independent neutrophil polarization. The proposed work is innovative because it utilizes a technique, recently developed by the applicant, to transfect primary human neutrophils. Our expectation is that this approach establish how IL8 and GROalpha regulate neutrophil polarization using two different mechanisms. This outcome is significant because it will suggest new targets for prevention and treatment strategies to control neutrophil recruitment in a variety of inflammatory diseases such as obliterative bronchiolitis, the most significant long-term complication in lung transplant patients. The proposed study fulfills the scope of the NIAID small research grants program as a small, self-contained research project requiring minimal funding for a limited time.